DESCRIPTION (APPLICANT'S ABSTRACT): The ends of mammalian chromosomes are protected by a nucleoprotein complex composed of TTAGGG repeats and associated proteins. This complex, the telomere, allows cells to make the distinction between random DNA breaks and natural chromosome ends, preventing inappropriate activation of DNA damage checkpoints and blocking unwarranted repair reactions. We have identified two proteins, TRF1 and TRF2, that bind to duplex TTAGGG repeats. Our preliminary data indicate that TRF1 and its interacting factor tankyrase are primarily involved in telomere length regulation while TRF2 is crucial for the protection of chromosome ends. TRF2 forms a complex that includes hRapl, the Mrel 1 recombination/repair complex, and the XPF/ERCC1 NER nuclease. Inhibition of TRF2 with a dominant negative allele resulted in immediate deprotection of telomeres leading to chromosome end fusions, loss of the ss telomeric overhang, induction of a p53/ATM-dependent DNA damage checkpoint, and apoptosis or senescence. Here we prospose to study the protection of mammalian telomeres by analysing the structure and function of the TRF2 complex. In AIM 1 we will verify the roles of TRF1 and TRF2 in telomere protection by analysing the null phenotype of both factors in genetically altered ES cells that allow conditional deletion of the TRF1 and TRF2 genes. The contribution of hRapl (AIM 2) and the Mrel 1 complex (AIM 3) to telomere protection will be similarly studied using dominant negative alleles and ES cells that are conditionally null for the pertinent genes. The interaction of the XPF/ERCC1 complex with TRF2 and telomeres will be studied and the role of this nuclease in telomere protection will be approached by analysing telomeres in the ERCC1 null mouse. The components of the TRF2 complex will be further defined (AIM 5) by nanoelectrospray tandem mass spectrometry of tryptic peptides of proteins associated with TRF2 and hRapl, by ChIP analysis of cross-linked telomeric chromatin, and by 2-hybrid screens for hRapl interacting factors. The results are expected to provide structural and functional insights in how chromosome ends are protected from fusion and degradation and how TRF2 prevents the activation of DNA damage checkpoints by telomere termini. Understanding how telomeres protect chromosome ends is important since telomere function has been implicated in human cancer.